In multicellular organisms, homeostasis is maintained by balancing the rate of cell proliferation against the rate of cell death. Cell proliferation is influenced by numerous growth factors and the expression of proto-oncogenes, which typically encourage progression through the cell cycle. In contrast, numerous events, including the expression of tumor suppressor genes, can lead to an arrest of cellular proliferation.
In differentiated cells, a particular type of cell death called apoptosis occurs when an internal suicide program is activated. This program can be initiated by a variety of external signals as well as signals that are generated within the cell in response to, for example, genetic damage. For many years, the magnitude of apoptotic cell death was not appreciated because the dying cells are quickly eliminated by phagocytes, without an inflammatory response.
The mechanisms that mediate apoptosis have been intensively studied. These mechanisms involve the activation of endogenous proteases, loss of mitochondrial function, and structural changes such as disruption of the cytoskeleton, cell shrinkage, membrane blebbing, and nuclear condensation due to degradation of DNA. The various signals that trigger apoptosis are thought to bring about these events by converging on a common cell death pathway that is regulated by the expression of genes that are highly conserved from worms, such as C. elegans, to humans. In fact, invertebrate model systems have been invaluable tools in identifying and characterizing the genes that control apoptosis. Through the study of invertebrates and more evolved animals, numerous genes that are associated with cell death have been identified, but the way in which their products interact to execute the apoptotic program is poorly understood.
Caspases, a class of proteins central to the apoptotic program, are responsible for the degradation of cellular proteins that leads to the morphological changes seen in cells undergoing apoptosis. Caspases (cysteinyl aspartate-specific proteinases) are cysteine proteases having specificity for aspartate at the substrate cleavage site. Generally, caspases are classified as either initiator caspases or effector caspases, both of which are zymogens that are activated by proteolysis that generates an active species. An effector caspase is activated by an initiator caspase which cleaves the effector caspase. Initiator caspases are activated by an autoproteolytic mechanism that is often dependent upon oligomerization directed by association of the caspase with an adapter molecule.
Apoptotic signaling is dependent on protein-protein interactions. At least three different protein-protein interaction domains, the death domain, the death effector domain and the caspase recruitment domain (CARD), have been identified within proteins involved in apoptosis. A fourth protein-protein interaction domain, the death recruiting domain (DRD) was recently identified in murine FLASH (Imai et al. (1999) Nature 398:777-85).
Caspases comprise a multi-gene family having at least 12 distinct family members (Nicholson (1999) Cell Death and Differentiation 6:1028). A relatively small fraction of cellular polypeptides (less than 200) are thought to serve as targets for cleavage by caspases. Because many of these caspase targets perform key cellular functions, their proteolysis is thought to account for the cellular and morphological events that occur during apoptosis. Members of the caspase gene family can be divided by phylogenetic analysis into two major subfamilies, based upon their relatedness to ICE (interleukin-1β converting enzyme; caspase-1) and CED-3. Alternate groupings of caspases can be made based upon their substrate specificities.
Many caspases and proteins that interact with caspases possess a CARD domain. Hofmann et al. ((1997) TIBS 22:155) and others have postulated that certain apoptotic proteins bind to each other via their CARD domains and that different subtypes of CARD domains may confer binding specificity, regulating the activity of various caspases, for example.
Apoptosis in mammalian cells is mediated by large protein families that share sequence and structural similarity with the core apoptotic proteins of Caenorhabditis elegans (Metzstein et al. (1998) Trends. Genet. 14:410). The nematode CED-4 protein and its human homolog Apaf-1 play central roles in apoptosis by transducing death signals to the activation of caspases. Both CED-4 and Apaf-1 contain an N-terminal CARD domain that mediates caspase binding and a centrally located nucleotide-binding site (NBS) domain. Unlike CED-4, Apaf-1 contains a C-terminal WD-40 domain that mediates protein activation in response to the release of mitochondrial cytochrome c (Zou et al. (1997) Cell 90:405; Li et al. (1997) Cell 91:479; Srinivasula et al. (1998) Mol. Cell. 1:949). Additional CED4/Apaf-1 family members include CARD-4, Nod2 and CARD-7 (NAC/DEFCAP) (Bertin et al. (1999) J. Biol. Chem. 274:12955; Bertin et al. (2000) J. Biol. Chem. 275:41082; Inohara et al. (2000) J. Biol. Chem. 275:27823; Chu et al. (2001) J. Biol. Chem. 276:9239; Hliang et al. (2001) J. Biol. Chem. 276:9230). CARD4, Nod2 and CARD7 each contain NBS domains and effector CARD domains that mediate binding to downstream CARD-containing signaling partners. Both CARD-4 and Nod2 assemble together with the CARD protein RICK and induce the activation of NF-kB. Recent evidence suggests that CARD-7 may play a role analogous to Apaf-1 and directly mediate caspase activation. In addition, each protein contains extensive leucine-rich repeats (LRR) that have been proposed to function as binding sites for upstream regulators. The structure of CARD-4, Nod2 and CARD-7 is strikingly similar to plant NBS/LRR proteins that induce gene expression and cell death in response to pathogen infection (Dixon et al. (2000) Proc. Nat'l. Acad. Sci. USA 97:8807). Thus, CARD-4, Nod2 and CARD-7 likely play critical roles in stress-activated signaling pathways and may be components of the host innate immune response.